Recombinant Mycobacterium avium Elongation factor Ts (tsf)

What is the functional role of Elongation Factor Ts in M. avium protein synthesis?

Elongation Factor Ts (Ef-Ts) in Mycobacterium avium functions as a guanine nucleotide exchange factor that catalyzes the regeneration of active Elongation Factor Tu (Ef-Tu). During protein synthesis, Ef-Tu binds GTP and delivers aminoacyl-tRNA to the ribosome. After GTP hydrolysis, Ef-Tu remains bound to GDP in an inactive state. Ef-Ts facilitates the exchange of GDP for GTP, recycling Ef-Tu for subsequent rounds of elongation . In mycobacteria, the tsf gene (homologous to rv2889c in M. tuberculosis) encodes Ef-Ts. This protein-protein interaction is critical for maintaining efficient protein synthesis, particularly under stress conditions that M. avium encounters during infection.

How does the structure of M. avium Ef-Ts compare with other mycobacterial species?

M. avium Ef-Ts shares significant structural homology with other mycobacterial Ef-Ts proteins, particularly with M. tuberculosis Ef-Ts. The protein contains conserved domains that mediate interaction with Ef-Tu, including regions that disrupt the Ef-Tu-GDP complex to facilitate nucleotide exchange. Analysis of sequence alignments reveals that while the core functional domains remain conserved across mycobacterial species, M. avium Ef-Ts may contain unique structural elements that potentially contribute to its adaptation to different host environments . These structural differences can be methodically investigated using comparative modeling approaches followed by molecular dynamics simulations to identify species-specific interaction sites.

How is the expression of M. avium tsf gene regulated during infection?

The expression of the M. avium tsf gene undergoes complex regulation during infection, similar to other mycobacterial translation factors. Research indicates that under stress conditions, including nutrient limitation and exposure to host immune factors, M. avium may modulate tsf expression to adapt protein synthesis rates. Methodologically, this can be studied through:

• RNA extraction from M. avium under various stress conditions

• qRT-PCR analysis targeting the tsf transcript

• Reporter gene assays using the tsf promoter region

• Chromatin immunoprecipitation to identify regulatory proteins

Studies with M. tuberculosis have shown that translation factors can be differentially regulated during infection phases, suggesting M. avium likely employs similar adaptations to persist within macrophages .

What are the optimal expression systems for producing recombinant M. avium Ef-Ts?

For recombinant expression of M. avium Ef-Ts, several expression systems have been evaluated, each with distinct advantages:

For functional studies, M. smegmatis expression is methodologically superior as it provides a more native-like environment, while E. coli systems are preferable for structural studies requiring larger protein quantities .

What purification protocols yield the highest functional activity of recombinant M. avium Ef-Ts?

A methodological approach to purifying functional M. avium Ef-Ts involves:

• Affinity chromatography: Using Ni²⁺-NTA resin for His-tagged protein with extensive washing using 20 mM imidazole to remove non-specifically bound proteins .

• Buffer optimization: Purification in 1× PBS with 1 mM PMSF, 10% glycerol, and elution with 500 mM imidazole .

• Desalting: Immediate removal of imidazole using PD-10 columns to prevent protein destabilization .

• Ion exchange chromatography: Further purification using a salt gradient on a MonoQ column at pH 8.0.

• Size exclusion chromatography: Final polishing step to ensure homogeneity and removal of aggregates.

This sequential purification approach minimizes the co-purification of E. coli or M. smegmatis proteins that could interfere with subsequent functional assays. Yield can be assessed using Bradford assay, while purity should be confirmed by SDS-PAGE with Coomassie staining (>95% purity required for functional studies) .

How can researchers validate the correct folding and activity of purified recombinant M. avium Ef-Ts?

Validation of correctly folded and functional recombinant M. avium Ef-Ts requires multiple complementary approaches:

• Circular dichroism spectroscopy to assess secondary structure elements

• Thermal shift assays to evaluate protein stability

• Size exclusion chromatography to confirm monomeric state

• Nucleotide exchange activity assay measuring the rate of GDP-to-GTP exchange on Ef-Tu

• Surface plasmon resonance to quantify binding kinetics with Ef-Tu

The definitive functional validation involves demonstrating that purified Ef-Ts accelerates the release of GDP from Ef-Tu and facilitates the formation of the Ef-Tu-GTP complex. This can be measured using fluorescently labeled GDP analogs or radiolabeled [³H]-GDP in a time-course dissociation assay .

How can researchers establish in vitro systems to study M. avium Ef-Ts and Ef-Tu interactions?

To establish robust in vitro systems for studying M. avium Ef-Ts and Ef-Tu interactions, researchers should:

• Co-express both proteins using dual expression vectors like pETDuet-1, with His₆-tagged Ef-Tu in MCS1 and MBP-tagged Ef-Ts in MCS2 .

• Implement pull-down assays using either Ni²⁺-NTA (for His-tagged protein) or amylose resin (for MBP-tagged protein) to confirm complex formation.

• Utilize isothermal titration calorimetry (ITC) to determine binding stoichiometry, affinity constants, and thermodynamic parameters.

• Employ fluorescence resonance energy transfer (FRET) by labeling Ef-Tu and Ef-Ts with appropriate fluorophores to monitor real-time interaction dynamics.

• Develop a reconstituted translation system using purified mycobacterial ribosomes, tRNAs, and aminoacyl-tRNA synthetases to assess the functional significance of these interactions.

These methodological approaches provide complementary data that together establish a comprehensive picture of how these two elongation factors cooperate in the mycobacterial translation machinery .

What experimental designs can elucidate the impact of post-translational modifications on M. avium Ef-Ts function?

To systematically investigate post-translational modifications (PTMs) of M. avium Ef-Ts:

• Phosphorylation analysis:

  • Express Ef-Ts in the presence of mycobacterial kinases (e.g., PknB) in dual expression systems

  • Analyze phosphorylation sites using LC-MS/MS after phosphopeptide enrichment

  • Create phosphomimetic mutants (Ser/Thr to Asp/Glu) and phosphoablative mutants (Ser/Thr to Ala)

  • Compare nucleotide exchange activity between wild-type, phosphorylated, and mutant proteins

• Other PTMs investigation:

  • Screen for acetylation and methylation using specific antibodies

  • Perform in vitro modification assays with mycobacterial lysates

  • Identify modified residues through mass spectrometry

• Functional consequences assessment:

  • Determine how PTMs affect Ef-Ts:Ef-Tu binding affinity using SPR

  • Measure GDP/GTP exchange rates with modified versus unmodified Ef-Ts

  • Assess impact on protein stability and half-life

Similar to M. tuberculosis Ef-Tu, which shows altered activity when phosphorylated by PknB, M. avium Ef-Ts likely undergoes regulatory PTMs that fine-tune translation in response to environmental conditions .

How should researchers design experiments to study the role of M. avium Ef-Ts in bacterial persistence?

To investigate M. avium Ef-Ts's role in bacterial persistence, researchers should employ:

• Conditional gene expression systems:

  • Develop tetracycline-inducible or repressible tsf expression constructs

  • Create merodiploid strains to study partial depletion effects

• Macrophage infection models:

  • Establish MPI cell infections as described for MAC studies

  • Monitor bacterial survival during Ef-Ts depletion/overexpression

  • Quantify protein synthesis rates using [³⁵S]-methionine incorporation

• Stress response analysis:

  • Subject M. avium with modified tsf expression to various stressors (nutrient limitation, acidic pH, oxidative stress)

  • Measure survival rates and protein synthesis capacity

  • Analyze stress-response gene expression patterns by RNA-seq

• Animal infection models:

  • Compare virulence of wild-type versus tsf-modified strains

  • Assess bacterial load in tissues over extended time periods

  • Evaluate histopathological changes in infected tissues

These methodological approaches can reveal whether modulation of Ef-Ts activity represents a potential vulnerability in M. avium persistence mechanisms, similar to the way PknB-mediated phosphorylation affects M. tuberculosis Ef-Tu function .

What are common challenges in establishing stable expression of recombinant M. avium Ef-Ts and how can they be resolved?

When expressing recombinant M. avium Ef-Ts, researchers commonly encounter these challenges and solutions:

Systematic optimization of these parameters is crucial, starting with small-scale expression tests before scaling up to preparative quantities .

How can researchers accurately quantify the nucleotide exchange activity of M. avium Ef-Ts?

For precise quantification of M. avium Ef-Ts nucleotide exchange activity:

• Direct measurement approach:

  • Pre-load M. avium Ef-Tu with [³H]-GDP or fluorescent mant-GDP

  • Add varying concentrations of Ef-Ts (0.1-10 μM)

  • Monitor nucleotide release rates using filter binding assays or fluorescence spectroscopy

  • Calculate kinetic parameters (kcat, KM) using Michaelis-Menten or similar models

• Coupled enzyme assay approach:

  • Link GTP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase

  • Monitor A340 decrease as measure of nucleotide exchange and subsequent GTP hydrolysis

  • Determine rate-limiting steps through systematic variation of component concentrations

• Real-time binding analysis:

  • Immobilize Ef-Tu-GDP on sensor chip surface

  • Flow solutions containing Ef-Ts and GTP

  • Measure association/dissociation rates through surface plasmon resonance

  • Derive kinetic constants from sensorgram analysis

These methodological approaches provide complementary data on the catalytic efficiency of Ef-Ts and allow comparison between wild-type and mutant proteins or between different mycobacterial species .

What statistical approaches should be used when comparing the effects of mutations on M. avium Ef-Ts function?

When analyzing mutational effects on M. avium Ef-Ts function, these statistical approaches are recommended:

• Experimental design considerations:

  • Minimum of 3-5 biological replicates per condition

  • Include positive and negative controls in each experiment

  • Randomize sample processing order to minimize bias

  • Use power analysis to determine appropriate sample sizes

• Data normalization strategies:

  • Normalize activity measurements to wild-type protein under identical conditions

  • Account for batch effects using mixed-effects models

  • Transform data when necessary to meet normality assumptions

• Statistical tests for comparing multiple mutations:

  • One-way ANOVA followed by Tukey's or Dunnett's post-hoc tests for multiple comparisons

  • Non-parametric alternatives (Kruskal-Wallis with Dunn's test) for non-normally distributed data

  • Bonferroni or Benjamini-Hochberg corrections to control for family-wise error rate

• Correlation analyses:

  • Multiple regression to identify relationships between biochemical parameters

  • Principal component analysis to reduce dimensionality when examining multiple functional parameters

  • Hierarchical clustering to identify functionally similar mutants

These methodological approaches ensure rigorous evaluation of whether observed differences between wild-type and mutant Ef-Ts proteins are statistically significant and biologically meaningful .

How can structural biology techniques advance our understanding of M. avium Ef-Ts function?

Advanced structural biology approaches offer powerful methodologies for elucidating M. avium Ef-Ts function:

• X-ray crystallography workflow:

  • Optimize purification to achieve >98% homogeneity and 10-15 mg/ml concentration

  • Screen 500-1000 crystallization conditions using sitting-drop vapor diffusion

  • Collect diffraction data at synchrotron radiation sources

  • Solve structure using molecular replacement with M. tuberculosis Ef-Ts as a search model

  • Analyze nucleotide binding sites and Ef-Tu interaction interfaces

• Cryo-electron microscopy approach:

  • Prepare Ef-Ts:Ef-Tu complexes in different nucleotide-bound states

  • Collect single-particle data on high-end microscopes with direct electron detectors

  • Perform 3D reconstruction to resolve conformational states during nucleotide exchange

  • Map functionally important residues onto the structure

• NMR spectroscopy for dynamics:

  • Produce ¹⁵N/¹³C-labeled Ef-Ts in minimal media

  • Collect HSQC spectra to monitor conformational changes upon Ef-Tu binding

  • Perform relaxation experiments to identify flexible regions important for function

These methodological approaches can reveal the structural basis for species-specific differences in Ef-Ts function and identify potential sites for targeted inhibitor design .

What approaches can identify small-molecule modulators of M. avium Ef-Ts activity?

To identify small-molecule modulators of M. avium Ef-Ts:

• High-throughput screening methodology:

  • Develop a fluorescence polarization assay using labeled GDP to monitor displacement from Ef-Tu

  • Screen compound libraries (10,000-100,000 compounds) in 384-well format

  • Implement Z' factor analysis to ensure assay robustness (Z' > 0.7)

  • Confirm hits with dose-response curves and orthogonal assays

• Structure-based design approach:

  • Identify binding pockets at the Ef-Ts:Ef-Tu interface through computational analysis

  • Perform virtual screening of compound libraries against these pockets

  • Synthesize or purchase top-scoring compounds for experimental validation

  • Optimize lead compounds through medicinal chemistry

• Fragment-based discovery:

  • Screen fragment libraries using thermal shift assays or STD-NMR

  • Link or grow promising fragments to improve potency

  • Validate binding modes using X-ray crystallography

These methodological approaches could identify compounds that modulate protein synthesis in M. avium, potentially offering new therapeutic strategies against MAC infections, similar to how kirromycin affects Ef-Tu function .

How can systems biology approaches integrate M. avium Ef-Ts function into broader cellular networks?

Systems biology methodologies for contextualizing M. avium Ef-Ts function include:

These systems-level approaches provide a comprehensive understanding of how Ef-Ts functions within the broader context of mycobacterial physiology and stress adaptation, revealing potential vulnerabilities that could be exploited in MAC infections .